Project Summary Intranasal oxytocin (OT) decreases alcohol craving and affective dysfunction in abstinent alcoholics, providing a potential pharmacotherapy for alcoholism. Excessive habitual ethanol use dysregulates mesocorticolimbic reward circuitry which may include decreased OT activation of neurons in the ventral tegmental area (VTA). Here, we propose that ethanol dysregulation of OT signaling specifically alters oxytocinergic innervation and OT receptor (OTR) expression within the VTA, which in turn, facilitates ethanol-seeking in an effort to maintain VTA activation. Recently, we discovered that VTA neurons that synthesize glutamate (GLU) and dopamine (DA) express OTRs indicating the complexity of OT regulation of VTA function and reward. Our use of advanced neuroanatomical techniques combined with in vivo optogenetics provides a unique opportunity to quantify ethanol-induced plasticity in OT signaling in the VTA as well as the consequences of this plasticity on reward seeking behavior. Specifically, we will use genetically altered mice, directed viral gene transfer, and in vivo optogenetics to identify and manipulate pre- and post-synaptic OT signaling in the VTA after daily binge ethanol intoxication. These studies will test the overall hypothesis that manipulations of pre- and post-synaptic OT signaling in the VTA alters reward states and that OT regulation of VTA circuitry and positive reinforcement is impaired by daily binge ethanol intoxication. Aim 1 will quantify the impact of daily binge ethanol intoxication on the number and phenotype of OTR-expressing neurons in the VTA by delivering Cre- inducible adenoassociated virus to mice that have Cre recombinase directed to the OTR gene (OTR-Cre). The effects of daily binge ethanol on hypothalamic OT mRNA and oxytocinergic innervation of the VTA will be quantified using amplified in situ hybridization and mice with Cre recombinase directed to the OT gene. Aim 2, will reveal functional consequences of ethanol-induced structural plasticity by evaluating how daily ethanol intoxication affects operant responding for stimulation of presynaptic or post-synaptic OT signaling within the VTA, which will be recapitulated using in vivo optogenetics. These experiments will determine whether daily binge ethanol decreases OT reward signaling by altering OT fibers and/or OTR expression by DA and GLU neurons in the VTA. Elucidating the impact of daily binge ethanol intake on endogenous OT regulation of VTA neurons will inform pathophysiologic mechanisms of alcohol use disorder and guide the development of novel therapeutic interventions for alcohol use disorders, particularly as they might differ according to sex.